Bacterial contamination is the major cause of food and water-borne infections in the world causing gastroenteritis, diarrhea, cramps, vomiting and often fever. In underdeveloped countries, these infections kill approximately 1.8 million people annually, most of whom are children. The Centers for Disease Control and Prevention (CDC) estimates that 76 million Americans become ill, more than 300,000 are hospitalized, and 5,000 people die from food-borne illnesses each year alone. After eating contaminated food, humans develop varying degrees of illness ranging from a short, mild gastrointestinal distress, such as that referred to as “food poisoning,” to life-threatening disease. The most commonly recognized food borne infections are those caused by bacteria such as, Salmonella, Listeria, Campylobacter, Staphylococcus aureus and E. coli O157:H7. For example, Salmonella are found throughout the environment, particularly in the intestinal tracts of birds, reptiles, farm animals and humans. Illness in humans often results from the eating of undercooked meats, milk or eggs or from cross-contamination of other foods which are eaten without cooking.
Harmful diseases such as cholera and shigellosis may be transmitted by contaminated water and are caused by bacteria which can often be traced to sources along rivers and lakes. Much of the contamination can be traced to leaking and overflowing sanitary sewer systems, wastewater treatment facilities, leachate from septic tanks, and fecal matter associated with storm runoff from areas with high densities of wildlife, pets or livestock. The principle bacterial pathogens that have been shown to cause human intestinal disease associated with drinking water are: Salmonella typhi (Typhoid fever) Salmonella paratyphi-A (paratyphoid fever); other Salmonella species (salmonellosis, enteric fever); Shigella dystenteriae, S. flexneri, and S. sonnei (bacillary dysentery); Vibrio cholerae (cholera); Leptospira spp., (leptospirosis); Yersinia enterocolitica (gastroenteristis); Francisella tularensis, (tularemia); Escherichia coli (gastroenteritis); and Pseudomonas aeruginosa, (various infections). Because of the seriousness of the diseases caused by water-borne bacteria and the importance of water as a natural resource, levels of “indicator” bacteria called coliform bacteria are often monitored. These indicator bacteria usually are harmless, but there are a few exceptions. Certain strains of E. coli have been associated with gastrointestinal infections in adults, known as traveler's diarrhea, urinary tract infections, and newborn meningitis. Certain strains of Klebseilla pneumonia have been associated with gastrointestinal infections, pneumonia, hospital-acquired urinary tract infections, burn wound infections, and as secondary invaders in other respiratory infections. Enterobacter has been associated with hospital acquired urinary tract infections. Citrobacter has been associated with urinary tract infections, superficial wound infections, osteomyelitis, neonatal meningitis, and gastroenteritis.
Similarly, bacterial contamination in medical or biological diagnostic methods presents a problematic and expensive obstacle to such diagnostics. Bacterial contamination in a biological sample such as blood, saliva or tissue culture can greatly impair a satisfactory result from many biological assays. Common approaches include treating biological samples with very high doses of antibiotics often without satisfactory results.
Industrial bacterial fermentation is another commercial area affected by bacterial contamination. Industrial production of ethanol fuel in the United States in 2005 is estimated to be 4.4 billion gallons, up two fold from the previous year. Ethanol is produced primarily using yeast (Saccharomyces) fermentation, with corn being the predominant feedstock. Other organisms used to a lesser extent for ethanol production include the bacteria Zymomonas mobilis. Other feed stocks include sugar cane, rice straw, barley and wheat waste, potato waste, wood waste, municipal waste, and beverage industry waste. With such materials serving as feedstock, it is not surprising that most fermentations take place in the presence of significant bacterial contamination. Lactobacillus are the major contaminants in ethanol production and their presence and resultant lactic acid production reduces yeast growth and ethanol yield.
Similarly, amino acids produced by fermentation are predominantly lysine and glutamic acid (1 million metric tons collectively), with lesser amounts of threonine and tryptophan. This represents a 3.5 billion dollar market worldwide. The primary organism used for production is Corynebacterium glutamicum. Feed stocks for this process include corn wastes combined with waste products high in nitrogen. Bacillus spp. make up a considerable amount of the contaminants that can occur, and being rapid growers, compete with the Corynebacterium for nutrients. Other bacterial fermentation products include metabolites, vitamins, antibiotics and enzymes all produced within microorganism cultures that are susceptible to bacterial contamination.
Conventional methods for the detection of bacterial contamination employ non-selective or selective bacterial culturing, or enrichment, followed by plating the cultures on selective media for verification of suspect colonies. This approach is time consuming and can take several days before results are obtained. Alternative methods, utilizing immunoassay or nucleic acid-based detection technologies, are more rapid. However, these methods still require an enrichment step for production of a detectable signal. The length of time needed for sufficient enrichment is dictated by the growth rate of the target bacteria in the sample.
Most high sensitivity bacterial detection and enumeration methods require an overnight incubation. In addition, these methods often suffer from a lack of sensitivity or specificity or require expensive equipment or considerable technical expertise to perform. One particular problem with these methods is that, during the enrichment step, nearly all bacteria in the culture enjoy enhanced growth. The presence of large numbers of non-target bacteria in the enrichment culture causes interference with detection of the target bacteria, resulting in lack of sensitivity, cross-reactivity, false negative and false positive results. Scientists have attempted to reduce this problem by adding antibiotics to the enrichment cultures. However, the presence of antibiotics in the enrichment media decreases the growth rate of the target bacteria, lengthens the amount of time required to perform the assay and fails to eliminate cross-reactivity.
More recently developed bacterial detection assays combine an enriched bacterial culture with a lytic bacteriophage, a virus that specifically infects the target bacteria. Lytic phages are among the simplest and most abundant organisms on earth. Phages infect respective host bacteria and inject phage DNA. Within infected bacteria, phage DNA is replicated and then incorporated into new viral particles made by the bacterial host. New phage particles are then released from their host bacterial cells via a process known as “lysis,” which kills the infected bacterial cell. Lysis allows subsequent phage infection of adjacent bacteria in a rapid, exponential pattern. In contrast to lytic phages, temperate phages bolster their bacterial host's virulence, resilience, and general capacity to proliferate. In the newly developed bacterial detection assays, lytic bacteriophage are labeled in some way and the presence of the label is detected when the phage infects target bacteria present in the sample. As with assays employing immunoassay or nucleic acid based detection systems, the assays utilizing bacteriophage as means for detecting target bacteria also suffer from the inability to deliver rapid, highly sensitive results caused by the length of time required to grow and enrich the target bacteria and the simultaneous expansion of non-target bacteria in the sample that interferes or cross-reacts with detection of the target bacteria.
Therefore, what is needed is a bacterial detection method that is highly sensitive for minute concentrations of a target bacteria in a sample, such as a toxic bacterial contaminant in food, water, environmental, medical, agricultural, veterinary, pharmaceutical or industrial fermentation preparations yet provides rapid detection without creating the opportunity for undesirable false positive and negative results, and bacterial production processes that enrich for the target microorganism and improve production yield.